Fluorescent substances or fluorogenic substances, such as those that are responsive to enzyme activity, have a variety of useful applications. Such substances have been used in biological assays, for example.
Enzyme activity in a biological sample, such as a cell, a cell extract, a tissue sample, a biological fluid, a whole organism, and/or the like, is often associated with cellular metabolism, disease state, the success of a genetic manipulation, the identity of a particular microorganism, and/or the like. The ability to detect enzyme activity in a sensitive and quantitative manner may be useful for any of a variety of applications, such as use in cell biology, disease diagnosis, identification of a biological toxin, drug screening, and/or the like, for example. One way to detect enzyme activity is through the use of a fluorogenic enzyme substrate, which is a generally nonfluorescent or only weakly fluorescent compound until it is enzymatically cleaved to release a highly fluorescent dye.
Traditionally, a fluorogenic enzyme substrate has been produced by covalently linking a functional group of a fluorescent dye to a substrate moiety molecule, where the substrate moiety molecule mimics the natural enzyme substrate and thus is recognized by the enzyme being investigated. In general, the functional group of the dye, which is typically either an aromatic primary amine or an aromatic hydroxy group, is an integral part of the chromophoric core structure of the dye, in which the presence of the functional group imparts a spectral property or spectral properties unique to the dye. When the functional group of the dye is covalently linked to a substrate moiety molecule, the functionality of the functional group is changed, resulting in a dramatic blue shift in the absorption and emission wavelengths of the dye and a concomitant reduction in the fluorescent quantum yield of the dye. In some cases, the covalent linkage between the functional group and the substrate moiety molecule of the dye results in a completely colorless and nonfluorescent enzyme substrate. In general, when the functional group of the dye is an aromatic primary amine group, the substrate moiety molecule is an amino acid or a peptide that is recognized by a peptidase. In general, when the functional group of the dye is a hydroxy group, the substrate moiety molecule may be any of a variety of substrate moiety molecules, such as a glycosidyl that is recognized by a glycosidase, a phosphoryl that is recognized by a phosphatase, an alkyl that is recognized by a cytochrome P450 enzyme, or an acyl that is recognized by an esterase, for example. Enzymatic hydrolysis of the fluorogenic enzyme substrate cleaves the bond between the dye and the substrate moiety molecule, thus regenerating the fluorescent dye at a rate proportional to the level of enzyme activity.
Fluorogenic enzyme substrates have been produced using any of a number of fluorescent dyes. For example, amine-containing dyes, such as rhodamine 110, 7-amino-4-methylcoumarin, and 7-amino-4-trifluoromethylcoumarin, for example, have been used for preparing fluorogenic peptidase substrates. Further by way of example, hydroxy-containing dyes, such as fluorescein, 7-hydroxy-4-methylcoumarin, and resorufin, for example, have been used for preparing fluorogenic enzyme substrates in which the enzyme cleavage site is a bond between an oxygen atom and the enzyme substrate moiety molecule. In Table 1 below, a list of a few dyes that have been used for constructing fluorogenic enzyme substrates is provided, along with identifications of the functional group associated with each dye, the substrate moiety molecule associated with each dye, the type of linkage between the functional group and the substrate moiety molecule associated with each dye, and the enzyme that corresponds to the fluorogenic enzyme substrate associated with each dye, merely by way of example.
TABLE 1Dyes, and Associated Functional Groups,Substrate Moiety Molecules, Linkages, and EnzymesSubstrateFunctionalMoietyDyeGroupMoleculeLinkageEnzymerhodamine 110amineamino acid oramide bondpeptidasepeptide7-amino-4-amineamino acid oramide bondpeptidasemethyl-peptidecoumarinFluoresceinhydroxycarboxylic acidester bondesteraseFluoresceinhydroxyβ-D-galactoseether bondβ-galactosidaseFluoresceinhydroxyα-D-glucoseether bondα-glucosidaseFluoresceinhydroxyβ-D-cellobioseether bondcellulaseFluoresceinhydroxyphosphatephosphoesterphosphatasebondResorufinhydroxyalkylether bondcytochromeP4507-hydroxy-4-hydroxysulfatesulfoesteraryl sulfatasemethyl-bondcoumarin
Fluorogenic enzyme substrates have also been designed based on the principle of fluorescence resonance energy transfer (FRET). Such FRET-based design has primarily been used for preparing a fluorogenic peptidase substrate in which the enzyme must bind to both sides of the cleavage site for the enzymatic hydrolysis to take place. A FRET-based peptidase substrate has one dye, called the fluorescence donor, attached to one end of the peptide, and another dye, called the fluorescence acceptor or the fluorescence quencher, attached to the other end of the peptide. Prior to the enzymatic cleavage of the substrate, the fluorescence of the donor is substantially quenched by the quencher as a result of the physical proximity of the donor and quencher. Following the enzymatic hydrolysis of the peptide, the donor and quencher are separated, releasing the fluorescence of the donor at a rate proportional to the level of enzyme activity. There are various examples of FRET-based peptidase substrates, such as the HIV protease substrate described by Wang et al., Tetrahedron Lett. 31, 6493 (1990), the renin substrate described by Paschalidou et al., Biochem. J. 382, 1031 (2004), and the HCV substrate by Taliani et al., Anal. Biochem. 240, 60 (1996), for example. All of these FRET-based enzyme substrates employ a blue fluorescent donor dye. A dye having a short wavelength, such as a blue fluorescent dye, for example, is in general not desirable.
A class of fluorogenic substrates for TEM-1 β-lactamase or Bla, which is a bacterial enzyme that catalyzes the breakdown of cephalosporins with high efficiency, has been a useful variation of fluorogenic peptidase substrates. The gene that encodes for Bla has been used as a reporter gene for studying gene expression in eukaryotic cells. Several fluorogenic Bla substrates have been developed for detecting the reporter enzyme in transfected living cells. One Bla fluorogenic substrate, called CCF2, is a FRET-based compound consisting of a donor 7-hydroxycoumarin linked via a cephalosporin to an acceptor fluorescein. CCF2 is green fluorescent due to FRET from the donor to the acceptor, but becomes blue fluorescent when hydrolysis of the cephalosporin ring structure causes the elimination of the fluorescein molecule. (Zlokarnik et al., Science 279, 84 (1998).) FRET-based Bla substrates usually have relatively large molecular weights and poor water solubility, both of which make the substrates difficult to apply to mammalian tissues or cells with thick walls, such as yeast or plant cells, for example. New fluorogenic Bla substrates have been developed by attaching only a single dye with a phenolic group to the 3′-position of a cephalosporin. (Gao et al., J. Am. Chem. Soc. 125, 11146 (2003).) Enzymatic hydrolysis of the substrate releases the dye, resulting in a fluorescence increase. These new enzyme substrates have relatively small molecular weights and thus readily enter cells. However, the enzymatically released dyes from these single-dye substrates usually lack the ability to be retained in the cells, making it difficult to identify enzyme-activity-specific cells.
Further development of fluorescent or fluorogenic substances or the making or the use thereof is desirable.